SVG Tutorial for spatialHeatmap

Maintainer: Jianhai Zhang (jzhan067@ucr.edu; zhang.jianhai@hotmail.com)

1 Summary

This R package spatialHeatmap is used for interactive visualisation as long as a data matrix and an associated SVG image are provided. This tutorial is specifically designed for how to make an SVG image and link it with the data matrix. The tutorial requires a png image, where regions can be painted by different colours, a data matrix, the SVG editor Inkscape, and optionally the image editor GIMP. The png image is the template to make the SVG image while the data matrix is used to fill different regions in the image. Inkscape is used to associate the SVG image with the data matrix and GIMP is optionally used to facilitate making the SVG image.

In the following, the tutorial is given with a gene expression matrix and an associated root tissue image in SVG format as an example, which is just a basic example for users to start. All the files used in this tutorial can be downloaded here (download an SVG image: click the image, right-click, select “Save image as…”; download a PNG image: click the image, click “Download”, right-click, select “Save image as…”; download a TXT file: click the file, click “Raw”, right-click, select “Save as…”.).

Users are advised to pay enough attention to the bold text below, because it can prevent common errors when creating the SVG images.

2 Procedure

2.1 Gene Expression Matrix

In the gene expression matrix, the row and column names are usually gene IDs and sample/conditions, respectively. The sample/condition names MUST be fomatted this way: a sample name is followed by double underscore then the condition, such as “stele__140mM_48h" (Geng et al. 2013), where stele is the sample and 140mM_48h is the condition. One column of meta data (e.g. gene annotation) can also be included in parallel with sample/condition at the end. In the column names of sample/condition and meta data, only letters, digits, single underscore, dots are allowed. Not all samples in the matrix necessarily need to be present in the svg image, and vice versa. Only samples present in the svg image are recognised and coloured. The example of partial gene expression matrix is shown below (Y. Xie 2014; Zhu 2019; Dowle and Srinivasan 2018).

2.2 Make SVG Images

2.2.1 Make Blank SVG Images by Drawing

If the contour in the png image is not clear, GIMP can generate low-quality SVG images, so in this case one can draw the blank SVG image with Inkscape by using the png as a template. Below is an example of drawing only two polygons.

1. Draw polygons
   Open the root png image (Mustroph et al. 2009) in Inkscape. The image can be zoomed by press “-” or “+” on the keyboard. Select the “Draw freeahnd lines (F6)” at the left tool bar. Left click once at the first corner of the polygon, and move to the second corner and double left click, and so on. Lastly, when drawing the last line click at the first corner to seal the polygon.
   

2. Align polygons
   Select “Edit path by nodes (F2)” from the left tool bar. Draw a large rectangle to select the whole sealed polygon and draw another large rectangle to select all nodes.
   
   Click the “Make selected nodes smooth” in the top tool bar. Drag the edges and handles to make the sealed polygon aligned with the template, then the first polygon is finished.
   

3. Make polygons with shapes
   Alternatively, the polygons can be made with rectangles. Click “Create rectangles and squares (F4)” at the left tool bar and draw a rectangle in the second template polygon. Select the rectangle and click “Object to Path” under the “Path” tab at the top, then the rectangle becomes an SVG path, which can be edited. Switch cursor to “Select and Transform Objects (F1)” and rotate the rectangle.

    Switch the cursor to “Edit path by nodes (F2)” and select all nodes of the rectangle. Click “Make selected nodes corner” at the top tool bar. Drag the edges and handles to overlay the rectangle path on the template, then the second polygon is finished.

In this root image there are many polygons, so it takes too much time to draw them individually. However, GIMP can be used to extract the polygons automatically.

2.2.2 Make Blank SVG Images with GIMP

If the png image contains many polygons and their contour is clear (e.g. the root image), GIMP can be used to automatically extract the polygons.

4. Open the root png image with GIMP. There are five coloured tissues (skyblue, green, red, yellow, blue). The skyblue is not present in the gene matrix while the green, red, yellow, blue correspond to epidermis, cortex, endodermis, stele in the gene matrix respectively. Enable the “Paths” panel under the “Windows” tab at the top. Right click and select “By Color” under “Select”.
   

5. Click on the skyblue tissue, then right click, select “To Path” under “Select”, and the paths of all skyblue tissues are extracted in the “Paths” panel.
   

6. Similarly, exrtact paths of other tissues. In the “Paths” panel set paths visible by enabling the eye symbol, then right click and merge visible paths. Right click and export merged paths, which is a blank SVG image.
   

2.2.3 Make Blank SVG Images with GIMP and Drawing

The drawing method creates accurate SVG images but it is time-consuming, while the GIMP method is faster but it can generate fused polygons. In this tutorial, the root png image has well-separated polygons and clear contours, so GIMP can produce accurate SVG images. Otherwise the resulting SVG images would have mixed and noise polygons. Considering the pros and cons of two methods, the good practice is to use GIMP to extract polygons first then use drawing method to refine it. Below is an example of an SVG image with fused polygons, which is generated by GIMP.

7. Place all SVG paths inside a layer
   Open the blank fused SVG image in Inkscape (the image can be zoomed by press “-” or “+” on the keyboard). Under the “Layers” tab at the top click “Layers”, the “Layers” panel will come out on the right, then click “+” at the bottom left corner of the panel to add “Layer 1”.
   
   Draw a rectangle over the fused SVG graph and cut. Then click on “Layer 1” and paste the fused SVG to “Layer 1” (make sure the “Layer 1” is unlocked by refering to the lock symbol). Open the “XML Editor” from the “Edit” tab at the top, if “<svg:path id=”path 77“>” is under “<svg:g id=”layer1" inkscape:label=“Layer 1”>“, then the fused SVG is inside "Layer 1" for sure.
   

8. Refine and modify the SVG image
   Draw a ractangle over the image, click “Break Apart” under the “Path” tag, then select the outer noisy rectangle by clicking on its edge. Press “delete” on the keyboard to delete it.
   
   Click the edge of the large fused polygon and move. Use the drawing method to make new polygons (blue) with the fused ones as templates. Place back the new polygons to make the tissue complete.

If a large fused polygon needs to be separated, one can use the eraser tool. Drag the fused polygon away from the tissue, select the eraser tool from the left tool bar, then use the eraser to cut the fused polygon into three independent polygons. Select the cut polygons and click “Break Apart” under the “Path” tab at the top, then the three polygons are entirely separated. Place back the three polygons to make the tissue complete.

9. Fill and stroke
   Under “Object” tab at the top, select “Fill and Stroke…”, then the “Fill and Stroke (Shift+Ctrl+F)” panel will come out on the right. Select all polygons by drawing a large rectangle over them.
   
   Under the “Stroke paint” tab in the fill and stroke panel, select “Flat color”. Must make sure the “Alpha (opacity)” is 255, “Opacity(%)” is 100.0, and “Blur(%)” is 0.0 . Under the “Stroke style” tab, set the stroke width, e.g.: 1.5 px.
   
   Under the “Fill” tab, click “No paint” to get a blank SVG image, which is ready to use in next section.
   

2.3 Link the SVG Image with Gene Matrix

In the SVG image, each polygon has a unique ID. To display spatial heatmaps, these IDs should be replaced with sample names exactly from gene matrix. No matter how the blank SVG image is created, it should be placed inside a layer before start the following steps .

10. Group same tissues
    If multiple polygons belong to the same tissue type, they can be grouped together. The example of grouping epidermis is given below. Open “XML Editor…” under the “Edit” tab on the top, then the “XML Editor (Shift+Ctrl+X)” panel comes out on the right. Select all the epidermis polygons while pressing the “Shift” key, right click and select “Group”.
    
    

11. Associate polygons with samples
    The corresponding epidermis group <svg:g id=“987”> shows up in the XML panel. Click “id”, change “g987” to “epidermis”, and click “Set”, then the new group id is set. This means all the polygons in this group correspond to epidermis.
    
    In the “Fill and Stroke (Shift+Ctrl+F)” panel, select “Flat color” under the “Fill” tag, then specify a color for epidermis (e.g. green). Must make sure the “Alpha (opacity)” is 255, “Opacity(%)” is 100.0, and “Blur(%)” is 0.0 .
    
    Group other tissues, set ids and colours.
    

12. Tiny polygons
    If the resolution of the template png image is poor, GIMP can produce tiny polygons. These tiny polygons can interfere with colour mapping in the spatial heatmap, i.e. some polygons in one tissue are mapped with the colour from the neighbour tissue. If this error arises, it suggests the presense of “tiny” polygons. One sulution is to delete these tiny polygons from related tissue groups in the Inkscape XML editor. These tiny polygons only interfere with the group below them, so if such tiny polygons exist only in one tissue group (e.g. the stele tissue group in the root center), another solution is to move the group containing tiny polygons at the bottom in the XML editor, then no group will be interfered with.

13. Encircling polygons
    If a polygon encircles other polygons and does not have a corresponding sample in the gene matrix, just a structure of the whole tissue, its “path” should be moved to the top over other “paths” in the “XML Editor”. Otherwise, it can hide other tissues. E.g. In the brain SVG image (anatomybodysystem.com 2017; epilepsyresearch 2017), the grey outer polygon (the path “rect5480”) encircling all other polygons is at the top over other “paths”, which serves as a base for other paths.
    

2.4 Add Text to Label Tissues

Users can add text to label tissues. Basically, the text is first typed in with the text tool and then the text object is coverted to paths. Next paths are added into the polygon group of target tissue and filled with the same tissue colour.

14. Creat text paths
    Select “Creat and select text objects (F8)” from the left tool bar, drag a text box, and type (epidermis). Click on the text object and convert it to path.

15. Text fill and stroke
    Click on the text paths and fill them with the same colour of target tissue using “Pick colors from image (F7)” from the left tool bar. In the “Fill and Stroke” panel, set the stroke style and make sure the “Alpha (opacity)” is 255, “Opacity(%)” is 100.0, and “Blur(%)” is 0.0 for both fill and stroke .
    

16. Add text paths to tissue group
    Click and cut the text, then double click the target tissue (epidermis, green) to enter the group.
    
    
    Paste the text anywhere and move/resize it, then right click the text and ungroup it.
    
    All the text paths are in the target tissue group (epidermis).
    

To add a pointer, draw a rectangle and convert it to path, fill it with the same style as the target tissue, then move, rotate and resize it.

Clicking anyone of other tissues will group the text, pointer, and epidermis together, which can be confirmed by dragging epidermis to see they are moving together.

Similarly, add text to other tissues.

17. Save the final SVG graph, which is ready to use in the spatialHeatmap. The name of saved SVG image can only consists of letters, digits, underscores. E.g. “root_cross_final.svg” is acceptable while “root_cross_final(copy).svg” will give errors.

2.5 Troubleshooting

The process of making SVG image and linking it with data matrix is error-prone, if errors arise in the Shiny app, such as some tissues are missing, then users are advised to double check the bold text above, which can prevent most common errors.

Reference

anatomybodysystem.com. 2017. “HEAD ANATOMY.” http://anatomybodysystem.com/lateral-view-of-the-brain-labeled/lateral-view-of-the-brain-labeled-brain-diagram-and-label-anatomy-body-list/.

Dowle, Matt, and Arun Srinivasan. 2018. Data.table: Extension of ‘Data.frame‘. https://CRAN.R-project.org/package=data.table.

epilepsyresearch. 2017. “The Hippocampus: What Is It?” https://www.epilepsyresearch.org.uk/the-hippocampus-what-is-it/.

Geng, Yu, Rui Wu, Choon Wei Wee, Fei Xie, Xueliang Wei, Penny Mei Yeen Chan, Cliff Tham, Lina Duan, and José R Dinneny. 2013. “A Spatio-Temporal Understanding of Growth Regulation During the Salt Stress Response in Arabidopsis.” Plant Cell 25 (6): 2132–54.

Mustroph, Angelika, M Eugenia Zanetti, Charles J H Jang, Hans E Holtan, Peter P Repetti, David W Galbraith, Thomas Girke, and Julia Bailey-Serres. 2009. “Profiling Translatomes of Discrete Cell Populations Resolves Altered Cellular Priorities During Hypoxia in Arabidopsis.” Proc Natl Acad Sci U S A 106 (44): 18843–8.

Xie, Yihui. 2014. “Knitr: A Comprehensive Tool for Reproducible Research in R.” In Implementing Reproducible Computational Research, edited by Victoria Stodden, Friedrich Leisch, and Roger D. Peng. Chapman; Hall/CRC. http://www.crcpress.com/product/isbn/9781466561595.

Zhu, Hao. 2019. KableExtra: Construct Complex Table with ’Kable’ and Pipe Syntax. https://CRAN.R-project.org/package=kableExtra.